Cystathionase activity and glutathione metabolism in redifferentiating rat hepatocyte primary cultures

Abstract

Capacity to incorporate methionine sulfur into glutathione as well as cystathionase activity were lost in cultured hepatocytes in a biphasic manner with 75% of the total capacity disappearing with a half-life of about 10.6 hr, the remainder with a half-life of greater than 20 hr. Nicotinamide, 25 mM, produced a single phase loss with a t 1/2 of approximately 21 hr for both transsulfuration and cystathionase activity. Loss of both methionine sulfur incorporation and cystathionase activity occurred in transferrin/sodium selenite-supplemented Williams Medium E (TS-HWME) with a t 1/2 of about 96 hr through 72 hr in culture. Addition of the cystathionase inhibitor, propargylglycine, blocked glutathione synthesis in TS-HWME cells through 48 hr in culture, while propargylglycine blocked glutathione synthesis only at 4 hr in HWME cultured cells. Further, the accumulation of gamma-glutamyl transpeptidase was delayed by 48 hr in TS-HWME versus unsupplemented medium. Variation in the transport of sulfur amino acids was also found to occur with culture age. The Km values for cysteine and methionine transport were found to be approximately 150 and 100 microM, respectively, and were unaffected by culture age or the presence of TS-HWME. However, the Vmax for transport of methionine declined from 0.29 to 0.012 nmol/min/mg protein over 48 hr in culture. In TS medium, the Vmax at 48 hr for methionine transport had only decreased to 0.20 nmol/min/mg protein and increased for cysteine transport to 0.17 nmol/min/mg protein. These data suggest that during the redifferentiation of hepatocytes in culture, transsulfuration is regulated by control of the flow of substrate through cystathionase and that cystathionase is regulated by alteration of enzyme activity or content. Variations in the rate of transport of precursor sulfur amino acids are also an important component of the regulation of the net glutathione status of the redifferentiating hepatocyte.

Expression of Bcl-2 Increases Intracellular Glutathione by Inhibiting Methionine-Dependent GSH Efflux

“…As neither U937, a monocytic of [35S] methionine in media containing 0.03 mM methionine. Cells were lysed in buffer (1% Triton X-100, 0.5% sodium deoxycholate, cell line, or HepG2 cells, a human hepatoma line, are 0.1% SDS, 1 mM AEBSF, 2 mg/ml pepsatin, 3 mg/ml leupeptin) for capable of transsulfuration (22)(23)(24), effects on GSH con-10 min at 4CЊ. The lysate was centrifuged at 12,0001g for 10 min tent must affected by inhibition of GSH efflux via efflux at 4CЊ.…”

Rat hepatocytes prepared without collagenase: Prolonged retention of differentiated characteristics in culture

“…Although T/S supplementation did support higher GSH levels in EDTA-prepared cells, only the 72 hr point was significantly different (p > 0.01). The decline in GSH synthesis in collagenase cells has been related to changes in sulfur amino acid transport and the activity of CNase, the enzyme controlling the flow of methionine sulfur into cysteine via the transsulfuration pathway (Meredith, 1987). Figure 4 cOmpares the activity of CNase in collagenase cells to EDTA-prepared hepatocytes, with and without T/S supplementation.…”

“…Loss of hormone-dependent metabolism has been documented (Crabb and Roepke, 1987). Dramatic changes have been noted in the isozyme distribution of the cytochromes P-450 (Steward et al, 1985), glutathione (GSH) content and synthesis (Meredith, 1986), sulfur amino acid transport and metabolism, including loss of the characteristically adult transsulfuration pathway (Meredith, 1987) and many other differentiated metabolic functions. Alteration of medium conditions has led to improved hepatocyte function in culture (Barnes and Sato, 1980), and supplementation by selenium and transferrin has extended the useful lifespan of monolayer culture (Meredith, 1986(Meredith, , 1987 for metabolism studies.…”

Glutathione and glutathione conjugate efflux from cultured liver cells

“…A major factor in these changes are in alterations in the mechanism of cysteine supply (Meredith and Williams, 1986;Meredith, 1986;Ahmad et al, 1987;Meredith, 1987). These studies show the importance of comparing normal adult liver cells to those containing GGT, as in preneoplastic foci or other liver cells more advanced toward the hepatoma state.…”

“…These studies do not supply any information about the long-term regulation of the process or the regulation of effiux under chronic GSH depleting conditions. Moreover, the experiments were done with hepatocytes at 24 h in culture, the time when GSH-dependent metabolism and GSH synthesis are at a greatly reduced level (Meredith, 1986;Meredith, 1987). Freedman et al (1989) found that copper resistant hepatoma cells have elevated intracellular GSH levels and a decreased GSH effiux rate.…”